Project Summary A major advance in the therapy of retinal vascular diseases, including wet age-related macular degeneration (AMD) and diabetic macular edema (DME), is the approval of vascular endothelial growth factor (VEGF) inhibitors. However, anti-VEGF therapy has limited efficacies and may exert side effects on healthy cells and vasculature. In Preliminary Studies we identified a previous unappreciated angiogenic ligand. Neutralizing antibody against this ligand alleviates diabetic retinal vascular leakage and laser- or Matrigel-induced choroid neovascularization (CNV) in mice with similar efficacies to the FDA-approved VEGF inhibitor aflibercept. Unlike conventional angiogenic factors, such as VEGF, this new ligand induces or exacerbates angiogenesis only in the disease conditions but not in normal vasculature. These findings imply that anti-angiogenesis therapy against this target may have minimal side effects on healthy vasculature or cells. We further developed neutralizing monoclonal antibody (mAb) to facilitate the translation of this novel therapy. A barrier to the translation is the lack of the knowledge of the receptor for this angiogenic ligand and its signaling pathway. We hypothesize that this ligand regulates angiogenesis via a G protein-coupled receptor (GPCR) or receptor tyrosine kinase (RTK). The rationale of this hypothesis is that this ligand in Preliminary Studies stimulated the phosphorylation of ERK1/2 kinases, which are mostly activated by GPCRs or RTKs. The objective of this project is to identify and verify its unknown receptor. The unknown receptor on endothelial cells will be identified by its differential binding to its ligand in the presence or absence of excessive neutralizing mAb, followed by ligand-receptor complex purification and quantitative proteomics analysis. The extracellular domain of identified receptor(s) will be tested as a decoy receptor to neutralize the ligand binding and functional activity. Ligand-derived receptor-binding peptides will be characterized as competitive receptor antagonists. Identification of the receptor will provide molecular insight into the ligand preferential stimulation of angiogenesis in disease conditions. Thus, this project will facilitate not only the bench-to-bedside translation of the newly-generated neutralizing mAb but also the development of receptor antagonists for novel anti-angiogenesis therapies of retinal neovascular diseases.